The rate-limiting enzyme for glutathione (GSH) synthesis is gamma-glutamylcysteine synthetase (GCS). Despite much study over the past two decades, the molecular and signaling mechanisms involved in regulation of GCS activity in cells are still poorly understood. Nutrient-induced regulation of GCS gene expression has not been reported except for preliminary work done in our laboratory over the past few years. Our long-term goal is to elucidate the mechanisms by which dietary sulfur amino acid or protein level induces changes in levels of the two subunits of GCS and changes in GCS activity state as well as the role of cellular cysteine status in this process. Specific objectives of this project are: (1) to determine the effect of cysteine supplementation on the expression of the modifier subunit of GCS and to determine the effect of cysteine supplementation on the ratio of the catalytic and modifier subunits in liver cells; (2) to determine the effect of dietary protein and sulfur amino acid supplementation on the expression of the modifier subunit of GCS and the ratio of the catalytic and modifier subunits of GCS in liver of intact rats; (3) to determine if the level of the GCS modifier subunit is limiting for GCS holoenzyme formation in HepG2 cells; (4) to determine the roles of GCS gene transcription and message stability in regulation of GCS subunit mRNA levels in response to cysteine concentration; (5) to determine if GCS subunit degradation rates are regulated; (6) to assess the ability of cysteine analogs (esp. cysteamine) to downregulate GCS subunit expression and/or GCS activity; (7) to determine if expression of GCS subunit genes and GCS activity are regulated in response to cysteine concentration vs. GSH concentration or thiol/disulfide redox state; and (8) to determine if transcription factor Nrf2 is necessary for activation of GCS subunit gene transcription. Capacity for GSH synthesis, availability of the precursor amino acid cysteine, and steady-state GSH levels all contribute to the body's ability to defend itself against oxidative or chemical stressors.